Relationship between RUNX1 and AXIN1 in ER-negative versus ER-positive Breast Cancer

RUNX1 plays opposing roles in breast cancer: a tumor suppressor in estrogen receptor-positive (ER⁺) disease and an oncogenic role in ER-negative (ER^?) tumors. Potentially mediating the former, we have recently reported that RUNX1 prevents estrogen-driven suppression of the mRNA encoding the tumor suppressor AXIN1. Accordingly, AXIN1 protein expression was diminished upon RUNX1 silencing in ER⁺ breast cancer cells and was positively correlated with AXIN1 protein expression across tumors with high levels of ER. Here we report the surprising observation that RUNX1 and AXIN1 proteins are strongly correlated in ER^? tumors as well. However, this correlation is not attributable to regulation of AXIN1 by RUNX1 or vice versa. The unexpected correlation between RUNX1, playing an oncogenic role in ER^? breast cancer, and AXIN1, a well-established tumor suppressor hub, may be related to a high ratio between the expression of variant 2 and variant 1 (v2/v1) of AXIN1 in ER^? compared with ER⁺ breast cancer. Although both isoforms are similarly regulated by RUNX1 in estrogen-stimulated ER⁺ breast cancer cells, the higher v2/v1 ratio in ER^? disease is expected to weaken the tumor suppressor activity of AXIN1 in these tumors.     

Cadherin-6 is a putative tumor suppressor and target of epigenetically dysregulated miR-429 in cholangiocarcinoma

Cholangiocarcinoma (CC) is a rare malignancy of the extrahepatic or intrahepatic biliary tract with an outstanding poor prognosis. Non-surgical therapeutic regimens result in minimally improved survival of CC patients. Global genomic analyses identified a few recurrently mutated genes, some of them in genes involved in epigenetic patterning. In a previous study, we demonstrated global DNA methylation changes in CC, indicating major contribution of epigenetic alterations to cholangiocarcinogenesis. Here, we aimed at the identification and characterization of CC-related, differentially methylated regions (DMRs) in potential microRNA promoters and of genes targeted by identified microRNAs. Twenty-seven hypermethylated and 13 hypomethylated potential promoter regions of microRNAs, known to be associated with cancer-related pathways like Wnt, ErbB, and PI3K-Akt signaling, were identified. Selected DMRs were confirmed in 2 independent patient cohorts. Inverse correlation between promoter methylation and expression suggested miR-129-2 and members of the miR-200 family (miR-200a, miR-200b, and miR-429) as novel tumor suppressors and oncomiRs, respectively, in CC. Tumor suppressor genes deleted in liver cancer 1 (DLC1), F-box/WD-repeat-containing protein 7 (FBXW7), and cadherin-6 (CDH6) were identified as presumed targets in CC. Tissue microarrays of a representative and well-characterized cohort of biliary tract cancers (n=212) displayed stepwise downregulation of CDH6 and association with poor patient outcome. Ectopic expression of CDH6 on the other hand, delayed growth in the CC cell lines EGI-1 and TFK-1, together suggesting a tumor suppressive function of CDH6. Our work represents a valuable repository for the study of epigenetically altered miRNAs in cholangiocarcinogenesis and novel putative, CC-related tumor suppressive miRNAs and oncomiRs.     

Expression and epigenetic regulation of angiogenesis-related factors during dormancy and recurrent growth of ovarian carcinoma

The initiation of angiogenesis can mark the transition from tumor dormancy to active growth and recurrence. Mechanisms that regulate recurrence in human cancers are poorly understood, in part because of the absence of relevant models. The induction of ARHI (DIRAS3) induces dormancy and autophagy in human ovarian cancer xenografts but produces autophagic cell death in culture. The addition of VEGF to cultures maintains the viability of dormant autophagic cancer cells, thereby permitting active growth when ARHI is downregulated, which mimics the “recurrence” of growth in xenografts. Two inducible ovarian cancer cell lines, SKOv3-ARHI and Hey-ARHI, were used. The expression level of angiogenesis factors was evaluated by real-time PCR, immunohistochemistry, immunocytochemistry and western blot; their epigenetic regulation was measured by bisulfite sequencing and chromatin immunoprecipitation. Six of the 15 angiogenesis factors were upregulated in dormant cancer cells (tissue inhibitor of metalloproteinases-3, TIMP3; thrombospondin-1, TSP1; angiopoietin-1; angiopoietin-2; angiopoietin-4; E-cadherin, CDH1). We found that TIMP3 and CDH1 expression was regulated epigenetically and was related inversely to the DNA methylation of their promoters in cell cultures and in xenografts. Increased H3K9 acetylation was associated with higher TIMP3 expression in dormant SKOv3-ARHI cells, while decreased H3K27me3 resulted in the upregulation of TIMP3 in dormant Hey-ARHI cells. Elevated CDH1 expression during dormancy was associated with an increase in both H3K4me3 and H3K9Ac in two cell lines. CpG demethylating agents and/or histone deacetylase inhibitors inhibited the re-growth of dormant cancer cells, which was associated with the re-expression of anti-angiogenic genes. The expression of the anti-angiogenic genes TIMP3 and CDH1 is elevated during dormancy and is reduced during the transition to active growth by changes in DNA methylation and histone modification.     

An integrated analysis to predict micro‐RNAs targeting both stemness and metastasis in breast cancer stem cells

Several evidences support the idea that a small population of tumour cells representing self‐renewal potential are involved in initiation, maintenance, metastasis, and outcomes of cancer therapy. Elucidation of microRNAs/genes regulatory networks activated in cancer stem cells (CSCs) is necessary for the identification of new targets for cancer therapy. The aim of the present study was to predict the miRNAs pattern, which can target both metastasis and self‐renewal pathways using integration of literature and data mining. For this purpose, mammospheres derived from MCF‐7, MDA‐MB231, and MDA‐MB468 were used as breast CSCs model. They had higher migration, invasion, and colony formation potential, with increasing in stemness‐ and EMT‐related genes expression. Our results determined that miR‐204, ‐200c, ‐34a, and ‐10b contemporarily could target both self‐renewal and EMT pathways. This core regulatory of miRNAs could increase the survival rate of breast invasive carcinoma via up‐regulation of OCT4, SOX2, KLF4, c‐MYC, NOTCH1, SNAI1, ZEB1, and CDH2 and down‐regulation of CDH1. The majority of those target genes were involved in the regulation of pluripotency, MAPK, WNT, Hedgehog, p53, and transforming growth factor β pathways. Hence, this study provides novel insights for targeting core regulatory of miRNAs in breast CSCs to target both self‐renewal and metastasis potential and eradication of breast cancer.     

Efficacy and epigenetic interactions of novel DNA hypomethylating agent guadecitabine (SGI-110) in preclinical models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a deadly malignancy characterized at the epigenetic level by global DNA hypomethylation and focal hypermethylation on the promoter of tumor suppressor genes. In most cases it develops on a background of liver steatohepatitis, fibrosis, and cirrhosis. Guadecitabine (SGI-110) is a second-generation hypomethylating agent, which inhibits DNA methyltransferases. Guadecitabine is formulated as a dinucleotide of decitabine and deoxyguanosine that is resistant to cytidine deaminase (CDA) degradation and results in prolonged in vivo exposure to decitabine following small volume subcutaneous administration of guadecitabine. Here we found that guadecitabine is an effective demethylating agent and is able to prevent HCC progression in pre-clinical models. In a xenograft HCC HepG2 model, guadecitabine impeded tumor growth and inhibited angiogenesis, while it could not prevent liver fibrosis and inflammation in a mouse model of steatohepatitis. Demethylating efficacy of guadecitabine on LINE-1 elements was found to be the highest 8 d post-infusion in blood samples of mice. Analysis of a panel of human HCC vs. normal tissue revealed a signature of hypermethylated tumor suppressor genes (CDKN1A, CDKN2A, DLEC1, E2F1, GSTP1, OPCML, E2F1, RASSF1, RUNX3, and SOCS1) as detected by methylation-specific PCR. A pronounced demethylating effect of guadecitabine was obtained also in the promoters of a subset of tumor suppressors genes (CDKN2A, DLEC1, and RUNX3) in HepG2 and Huh-7 HCC cells. Finally, we analyzed the role of macroH2A1, a variant of histone H2A, an oncogene upregulated in human cirrhosis/HCC that synergizes with DNA methylation in suppressing tumor suppressor genes, and it prevents the inhibition of cell growth triggered by decitabine in HCC cells. Guadecitabine, in contrast to decitabine, blocked growth in HCC cells overexpressing macroH2A1 histones and with high CDA levels, despite being unable to fully demethylate CDKN2A, RUNX3, and DLEC1 promoters altered by macroH2A1. Collectively, our findings in human and mice models reveal novel epigenetic anti-HCC effects of guadecitabine, which might be effective specifically in advanced states of the disease.     

CpG island hypermethylation of tumor-suppressor genes in H. pylori-infected non-neoplastic gastric mucosa is linked with gastric cancer risk

Background and aim: Gastric carcinogenesis involves CpG island hypermethylation (CIHM) of tumor‐suppressor genes. Although the CIHM of these genes occurs in non‐neoplastic gastric cells, it is unclear whether this epigenetic alteration is linked with aging and/or gastric cancer risk. We investigated this linkage in noncancerous gastric mucosa infected with H. pylori. Subjects and methods: Noncancerous corpus mucosa was endoscopically obtained from H. pylori‐positive gastric cancer patients (n = 34), and age‐matched H. pylori‐positive noncancerous controls (n = 68). Genomic DNA retrieved from the mucosa was subjected to methylation‐specific polymerase chain reaction for p16, Ecad, and DAPK genes. Linkage between CIHM and clinicopathologic factors was evaluated. Results: CIHM rates of DAPK, Ecad, and p16 promoters were significantly higher in noncancerous gastric mucosa of gastric cancer patients (91, 88, and 68%, respectively) than in noncancerous controls (71, 53, and 25%, respectively). Multivariate regression analysis showed a significant linkage between CIHM in noncancerous mucosa and coexistence of gastric cancer. Significant linkage between polymorphoneutrophil infiltration and CIHM was observed except for CIHM of p16. No linkage was observed between CIHM and other parameters, including age. High CIHM status (all three tested genes methylated) was associated with an increased risk of gastric cancer, with an odds ratio of 9.8 (95% confidence interval, 3.8–25.3). Conclusions: In a subset of the H. pylori‐infected population, CIHM of tumor‐suppressor genes in noncancerous gastric mucosa is linked with the risk of gastric cancer and polymorphoneutrophil infiltration, but not aging. CIHM is a potential marker of gastric cancer risk.     

Cancer‐specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis

Wild‐type p53 functions as a tumour suppressor while mutant p53 possesses oncogenic potential. Until now it remains unclear how a single mutation can transform p53 into a functionally distinct gene harbouring a new set of original cellular roles. Here we show that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated full‐length p53 mRNA. Cells expressing mutant p53 exhibit “gain‐of‐function” cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Interestingly, Δ160p53‐overexpressing cells behave in a similar manner. In contrast, an exogenous or endogenous mutant p53 that fails to express Δ160p53 due to specific mutations or antisense knock‐down loses pro‐oncogenic potential. Our data support a model in which “gain‐of‐function” phenotypes induced by p53 mutations depend on the shorter p53 isoforms. As a conserved wild‐type isoform, Δ160p53 has evolved during millions of years. We thus provide a rational explanation for the origin of the tumour‐promoting functions of p53 mutations.     

Association of low-dose exposure to persistent organic pollutants with E-cadherin promoter methylation in healthy Koreans

Background: Persistent organic pollutants (POPs), despite their considerably low levels in humans, are an increasing concern for the general populations given their various adverse health problems, including metabolic and carcinogenic effects. DNA methylation deregulation is thought to be a key mechanism in the development of human chronic diseases including cancer.

Methods: In an attempt to identify biomarkers monitoring low-dose exposure and hazard, we explored whether organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) may influence the methylation of tumour suppressor gene E-cadherin (CDH1) using peripheral blood cells from 364 healthy Korean subjects.

Results: CDH1 methylation was observed in 78.3% of study subjects. Serum concentrations of OCPs or PCBs compounds were higher in CDH1 methylation-positive subjects than in methylation-negative ones. After adjusting for various covariates, the odds ratio of CDH1 methylation of the summary measure of PCBs were 1.0, 2.5 (95% confidence interval: 1.2–5.3), 3.6 (1.6–8.1), 3.6 (1.4–8.6), and 2.5 (1.1–5.7) across quintiles of PCBs (P_trend?=?0.01). The values of OCPs were 1.0, 0.9, 1.2, 2.4 (1.0–5.9), and 1.7 (P_trend?=?0.05).

Conclusions: In this exploratory study with a small sample, CDH1 methylation might be served as the epigenetic biomarker associated with POPs exposure and adverse health effect.